WO2003064672A1 - Extrait cellulaire pour synthese de proteine acellulaire et procede de production de cet extrait - Google Patents

Extrait cellulaire pour synthese de proteine acellulaire et procede de production de cet extrait Download PDF

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Publication number
WO2003064672A1
WO2003064672A1 PCT/JP2003/000975 JP0300975W WO03064672A1 WO 2003064672 A1 WO2003064672 A1 WO 2003064672A1 JP 0300975 W JP0300975 W JP 0300975W WO 03064672 A1 WO03064672 A1 WO 03064672A1
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protein synthesis
cell
extract
dialysis
cell extract
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PCT/JP2003/000975
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English (en)
Japanese (ja)
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Yaeta Endo
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Yaeta Endo
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Priority to EP03734897A priority Critical patent/EP1479776A4/fr
Priority to US10/503,259 priority patent/US8734856B2/en
Priority to JP2003564263A priority patent/JP3675804B2/ja
Priority to CA002483332A priority patent/CA2483332A1/fr
Publication of WO2003064672A1 publication Critical patent/WO2003064672A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the present invention relates to a cell extract used for cell-free protein synthesis with enhanced protein synthesis reaction activity, a method for producing the same, and the like.
  • a new protein synthesis method that overcomes these problems is a cell-free protein synthesis method that employs chemical methods to maximize the superior properties of living organisms.
  • a translation system for the genetic information of a living body is provided in an artificial container, and the system is designed to reconstruct a system that can incorporate desired amino acids, including unnatural types, using the synthesized nucleic acid as type II. It is. Since this system is not restricted by living organisms, it is expected that the number of protein species that can be synthesized will be almost infinite.
  • the yield of wheat germ extract prepared by such a method is not always sufficient depending on the target protein (for example, DNA binding protein such as transcription factor) due to the influence of other inhibitors. There was a problem such as not being able to obtain.
  • a conventional cell extract for cell-free protein synthesis has a problem in storage stability when a solution containing amino acids, energy sources, ions, etc. necessary for protein synthesis is added. Therefore, the cell extract and the solution containing the energy source and the like are provided separately, and the experimenter has to mix them with the translation type ⁇ every time the experiment is performed. In addition, the operation must be performed at a low temperature, which complicates the entire experimental operation and often causes a failure of protein synthesis.
  • such a method for providing a reagent for a cell-free protein synthesis reaction is not suitable for exhaustive synthesis of proteins from a large number of genes, and such complexity and shortcomings are required for future robotization. Was the biggest issue that had to be resolved.
  • the present invention provides a cell extract in which the protein synthesis activity is promoted in order to improve the complexity of the steps or the low protein synthesis activity of interest in a cell-free protein synthesis reaction. Furthermore, the synthesis of wheat embryo cell-free protein does not require the preparation of a reaction mixture, but only by adding the desired translation type (mRNA). It is an object of the present invention to provide a cell extract having high storage stability (that is, a ready-made cell extract) that enables simple and efficient protein synthesis and mass synthesis.
  • the present inventor has found that a wheat germ extract is eliminated, and dialysis is carried out using a regenerated cellulose membrane having a molecular weight of about 12,200 to 14,000 daltons. As a result, it was found that when low molecular substances were removed from the extract, the protein synthesis activity of the cell extract was remarkably promoted. Furthermore, when the low-molecular substances were eliminated by a dialysis method in a solution containing all of the components necessary for cell-free protein synthesis except for the translated type I, the cell extract after the treatment was converted to a conventional cell extract. The protein synthesis activity was promoted more than the extract. In addition, the protein synthesis activity did not decrease even after storage at 180 ° C. for 4 weeks, indicating that this could be used as a ready-made cell extract.
  • the present invention has been achieved based on these findings.
  • a cell extract which has a protein synthesis activity and does not contain a low-molecular-weight protein synthesis inhibitor
  • the low-molecular-weight protein synthesis inhibitor has an excluded molecular weight of 12,000 to 14,0,0
  • the cell extract according to the above (1) which can be removed by dialysis using a regenerated cellulose membrane of 00 Dalton.
  • (6) a method for producing a cell extract for cell-free protein synthesis, which comprises removing a low-molecular-weight protein synthesis inhibitor from a cell extract having protein synthesis activity;
  • the low-molecular-weight protein synthesis inhibitor is a protein synthesis inhibitor having a molecular weight of 14,000 daltons or less.
  • a kit for performing cell-free protein synthesis comprising at least the cell extract according to any one of (1) to (5) and (1′4) above,
  • FIG. 1 is a diagram showing the generation of insoluble substances by dialysis of a wheat germ extract and its inhibitory action by ATP.
  • FIG. 2 is an electrophoretic image showing the results of protein analysis by SDS-polyacrylamide gel electrophoresis of soluble and insoluble proteins in a dialysis membrane in the absence and presence of ATP.
  • FIG. 3 is a diagram showing the protein synthesis activity of a cell extract (after dialysis treatment) obtained by dialysis in the absence or presence of ATP.
  • Figure 4 shows a graph showing the results of quantification and qualitative analysis of nucleic acid components, particularly RNA, in the insoluble substance generated by dialysis, and an electrophoresis image.
  • FIG. 5 is a diagram showing the quantification of nucleic acid components (RNA) in the insoluble substance after the dialysis treatment and the protein synthesis activity of the soluble fraction.
  • FIG. 6 is a diagram showing the effect of the addition of a stabilizing component by a batch method on protein synthesis activity.
  • FIG. 7 is a diagram showing the temperature dependence of the protein synthesis promoting effect by dialysis of a cell extract.
  • FIG. 8 is a diagram showing the protein synthesis inhibitory activity of a substance contained in wheat germ and fractionated by a Sephadex G25 column.
  • FIG. 9 shows the performance of a ready-made wheat germ-derived cell extract and an electrophoresis image. '
  • the cell extract used for preparing the cell extract for cell-free protein synthesis of the present invention may be any cell extract having a protein synthesis ability in a cell-free protein synthesis system.
  • the cell-free protein synthesis system means that a component including ribosomes, which is a protein translation device provided in a cell, is extracted from an organism, and the extracted or transcribed or translated ⁇ type, nucleic acid, amino acid, This method is performed in a test tube by adding an energy source, various ions, a buffer, and other effective factors.
  • cell-free translation system those that use RNA as type I (hereinafter sometimes referred to as “cell-free translation system”) and those that use DNA to perform the reaction by further adding an enzyme necessary for transcription such as RNA polymerase ( This is hereinafter sometimes referred to as “cell-free transcription Z translation system”).
  • cell-free protein synthesis system in the present invention includes any of the above-described cell-free translation system and cell-free transcription / translation system.
  • cell extract used in the present invention include known ones such as Escherichia coli, plant seed germ, and cell extract of egret reticulocytes. These may be commercially available ones, or a method known per se, specifically, an E. coli extract may be prepared by the method described in Pratt, JMeta 1., Transcription and Translation, Hames, 179-209, BD & H iggins, S.J., eds, IRLP ress, Ox for d. (1 984).
  • Examples of commercially available cell extracts include those derived from Escherichia coli, such as E.co1iS30 extractsystem (Promega) and RTS 500 R aid Transcription System (Roche). Rabbit Reticulocyte Ly sate System (produced by Promega), and those derived from wheat germ include PROTE IOS TM (produced by TOYOBO).
  • the plant seed is preferably a plant belonging to the family Poaceae such as wheat, oats, rice and corn.
  • a cell extract using a wheat germ extract is preferable.
  • a method for preparing a wheat germ extract for example, Johnston, FB eta 1., Nature, 179, 160-161 (1957), or Erickson, AH et al., (1996) Meth. In En Zymol., 96, 38-50, etc. can be used, and will be described in more detail below.
  • the germ portion is very small, so that it is preferable to remove the portion other than the germ as much as possible in order to obtain the germ efficiently.
  • a mechanical force is applied to a plant seed to obtain a mixture containing embryos, endosperm crushed material, and seed coat crushed material, and the embryo crushed material, seed coat crushed material, etc. are removed from the mixture to obtain a crude mixture.
  • An embryo fraction (a mixture containing crushed endosperm and crushed seed coat, with germ as the main component) is obtained.
  • the force applied to the plant seed should be strong enough to separate the germ from the plant seed.
  • a mixture containing an embryo, a crushed endosperm, and a crushed seed coat is obtained by crushing a plant seed using a known crushing device.
  • the pulverization of the plant seeds can be carried out using a generally known pulverizing apparatus.
  • a pulverizing apparatus such as a pin mill or a hammer mill, which applies an impact force to an object to be pulverized.
  • the degree of pulverization may be appropriately selected depending on the size of the plant seed germ to be used.For example, in the case of wheat seed, the maximum length is usually 4 mm or less, preferably 2 mm or less. Crush into pieces.
  • the pulverization is preferably performed in a dry manner.
  • a crude germ fraction is obtained from the obtained milled plant seed using a generally known classification device, for example, a sieve.
  • a sieve for example, for wheat seeds, the mesh size is usually 0.5mn! ⁇ 2 Omm, preferably 0.7mm ⁇ l.4mm crude embryo fraction Get.
  • the seed coat, endosperm, trash, etc. contained in the obtained crude germ fraction may be removed by using wind power or electrostatic force.
  • a crude germ fraction can also be obtained by a method utilizing the difference in specific gravity between the germ, the seed coat and the endosperm, for example, by heavy liquid sorting.
  • a crude embryo fraction containing more embryos a plurality of the above methods may be combined. Further, embryos are selected from the obtained crude embryo fraction using, for example, a visual color selector.
  • the embryo fraction thus obtained may have endosperm components attached thereto, it is usually preferable to further carry out a washing treatment to purify the embryo.
  • the washing treatment is usually performed by dispersing and suspending the embryo fraction in water or an aqueous solution or an aqueous solution containing a surfactant cooled to 10 ° C or less, preferably 4 ° C or less, and the washing solution becomes cloudy. It is preferable to wash until it disappears. Further, it is more preferable that the embryo fraction is dispersed and suspended in an aqueous solution containing a surfactant at a temperature of usually 10 ° C. or less, preferably 4 ° C. or less, and washing is performed until the washing solution does not become cloudy. .
  • a nonionic surfactant is preferable.
  • the surfactant is a nonionic surfactant, it can be widely used.
  • polyoxyethylene derivatives such as bridge (Brij), triton (Triton), nonidet (Nonidet) P40, and Tween. Is exemplified.
  • Nonidet (Nonidet) P40 is the most suitable.
  • These nonionic surfactants can be used at a concentration sufficient to remove the endosperm component and do not adversely affect the protein synthesis activity of the germ component, but can be used, for example, at a concentration of 0.5%.
  • the cleaning treatment with water or an aqueous solution and the cleaning treatment with a surfactant may be performed on either one or both. Further, these cleaning treatments may be performed in combination with ultrasonic treatment.
  • the intact (germinating) germ obtained by washing the plant germ after selecting the plant germ from the crushed product obtained by crushing the plant seed as described above (preferably in the presence of an extraction solvent) After fragmentation, the resulting wheat germ extract is separated and further purified to obtain a wheat germ extract for cell-free protein synthesis.
  • Extraction solvents include buffer, potassium ion, Alternatively, an aqueous solution containing a thiol group antioxidant can be used. If necessary, chromium ions, L-type amino acids and the like may be further added. For example, a solution containing ⁇ -2-hydroxyethylpiperazine ⁇ , -2-ethanesulfonic acid ( ⁇ S), potassium acetate, magnesium acetate, L-amino acid and ⁇ or dithiothreitol, or Patterson et al.
  • a solution obtained by partially modifying the above method (a solution containing HEPE S-KOH, acetate lime, magnesium acetate, calcium chloride, L-amino acid and / or dithiothreitol) can be used as an extraction solvent.
  • the composition and concentration of each component in the extraction solvent are known per se, and those used in a method for producing a wheat germ extract for cell-free protein synthesis may be used. ⁇
  • the germ is mixed with an extraction solvent in an amount necessary for extraction, and the germ is subdivided in the presence of the extraction solvent.
  • the amount of the extraction solvent is usually 0.1 milliliter or more, preferably 0.5 milliliter or more, more preferably 1 milliliter or more, based on 1 g of the embryo before washing.
  • the upper limit of the amount of the extraction solvent is not particularly limited, it is usually 10 ml or less, preferably 5 ml or less, based on 1 g of the embryo before washing.
  • Embryos to be subdivided may be those that have been frozen as in the past, or those that have not been frozen, but those that have not been frozen are more preferred.
  • the device and method that can be used for the subdivision are not particularly limited as long as the above conditions are satisfied.
  • a device having a high-speed rotating blade like a warlinda blender is used.
  • the rotation speed of the blade is usually 1 It is at least 4,000 rpm, preferably at least 5,000 rpm, and is usually at most 3,000 rpm, preferably at most 2,500 rpm.
  • the rotation time of the blade is usually 5 seconds or more, preferably 10 seconds or more.
  • the upper limit of the rotation time is not particularly limited, but is usually 10 minutes or less, preferably 5 minutes or less.
  • the temperature for subdivision is preferably within a range where the operation can be performed at 10 ° C or lower, and particularly preferably about 4 ° C.
  • the conventional process of crushing the plant germ and the process of mixing the crushed plant germ and the extraction solvent to obtain a wheat germ extract can be performed simultaneously as one process.
  • a wheat germ extract can be obtained efficiently.
  • the above method may be referred to as “blender method”.
  • the wheat germ extract can be collected by centrifugation or the like and purified by gel filtration or the like to obtain a wheat germ extract.
  • the gel filtration can be performed, for example, using a gel filtration device which has been equilibrated with an appropriate solution in advance.
  • the composition and concentration of each component in the gel filtration solution are known per se, and are used for the production of wheat germ extracts for cell-free protein synthesis (eg, HEPES-KOH, potassium acetate, magnesium acetate, Dithiothreitol or a solvent containing an L-type amino acid).
  • the cell extract obtained in this way has RNAse activity and host Phatase activity is extremely reduced.
  • Microorganisms especially spores such as filamentous fungi, may be mixed in the embryo extract-containing liquid after gel filtration, and it is preferable to exclude these microorganisms. It is important to prevent microbial growth, especially during long-term (1 day or more) cell-free protein synthesis reactions.
  • the means for eliminating microorganisms is not particularly limited, but it is preferable to use a filtration sterilization filter.
  • the pore size of the filter is not particularly limited as long as microorganisms that may be contaminated can be removed. Power is usually 0.1 to 1 micrometer, preferably 0.2 to 0.5 micrometer. is there.
  • the size of the spores of a small class of Bacillus subtilis is 0.5 ⁇ X 1 ⁇ , so it is best to use a 0.2 ⁇ m filter (for example, Minisart TM manufactured by Sartorius). It is also effective for the removal of water.
  • a filter having a large pore size it is preferable to first perform filtration using a filter having a large pore size, and then use a filter having a pore size capable of removing microorganisms that may be contaminated.
  • the cell extract obtained in this manner contains substances that suppress the protein synthesis function contained or retained by the raw material cells (mRNA, tRNA, such as tritin, thionine, and ribonuclease; translation protein factors and ribosomes).
  • the endosperm containing the substance that acts on and inhibits its function is almost completely removed and purified.
  • the expression that the endosperm is almost completely removed and purified refers to a wheat germ extract from which the endosperm has been removed to the extent that ribosomes are not substantially deadenylated.
  • the degree to which no deadenination is performed means that the deadenination rate of the ribosome is less than 7%, preferably 1% or less.
  • Such a cell extract contains a small amount of a low-molecular-weight protein synthesis inhibitor (hereinafter, this may be referred to as a “low-molecular-weight synthesis inhibitor”) even when the above-mentioned preferred fragmentation method is used. Therefore, these low-molecular-weight synthesis inhibitors are fractionated and eliminated from the components of the cell extract depending on the difference in molecular weight.
  • the molecular weight of the substances to be eliminated is required for protein synthesis contained in cell extracts.
  • the factor may be smaller than the factor, but may vary depending on the method used to eliminate the small molecule synthesis inhibitor as described later.
  • the molecular weight of the substance eliminated by the dialysis is specific. Has a molecular weight of 50,000 to 14,000 daltons or less, preferably 14,000 daltons or less.
  • a method for eliminating a low-molecular-weight synthesis inhibitor from a cell extract a method known per se is used, and specifically, a method using dialysis through a dialysis membrane, a gel filtration method, or an ultrafiltration method Examples include a filtration method.
  • the term "free of a low-molecular-weight synthesis inhibitor” refers to "not containing" a low-molecular-weight synthesis inhibitor up to about the same level as the treatment for eliminating the low-molecular-weight synthesis inhibitor by the various methods described above. Is not contained, and whether or not it has been excluded can be confirmed by the level of its protein synthesizing activity of the obtained cell extract.
  • a case where the dialysis method is used will be described in detail.
  • dialysis membrane used for dialysis examples include those having an exclusion molecular weight of 5,000 to 12,000 daltons. Specifically, a regenerated cellulose membrane having an exclusion molecular weight of 12,000 to 14,000 daltons ( Viskase Sales, manufactured by Chicago) and Spectra / Pore 6 having an excluded molecular weight of 50, 00.0 (manufactured by SPECTRUM L ABOTRATOR IESINC., CA, USA) are preferably used. An appropriate amount of the above-mentioned cell extract is put into such a dialysis membrane, and dialysis is performed using a conventional method. The dialysis time is preferably about 30 minutes to 24 hours.
  • the insoluble material Means that the cell extract in the step of eliminating the small molecule synthesis inhibitor is subjected to appropriate conditions, specifically, centrifugation, filtration, etc., especially 100,000 to 80, OOOX g, preferably 30, The substance is collected as a precipitate by centrifugation at about 0000 Xg for about 5 to 60 minutes, preferably about 20 minutes.
  • a solution containing at least a high-energy phosphate compound such as ATP or GTP
  • ATP is preferably used as the high energy phosphate compound. It is also preferably performed in a solution containing ATP and GTP, more preferably ATP, GTP, and 20 amino acids.
  • stabilizing components When eliminating a low-molecular-weight synthesis inhibitor in a solution containing these components (hereinafter sometimes referred to as “stabilizing components”), add the stabilizing components to the cell extract in advance and incubate After that, this may be subjected to a step of eliminating a low-molecular-weight synthesis inhibitor.
  • dialysis is used to eliminate low-molecular-weight synthesis inhibitors
  • low-molecular-weight synthesis inhibitors can be eliminated by adding dialysis by adding stabilizing components not only to cell extracts but also to external dialysis solutions. . It is more preferable to add a stabilizing component to the external dialysis solution, since a new stabilizing component is always supplied even if the stabilizing component is decomposed during dialysis.
  • the amount of the stabilizing component added and the stabilization time can be appropriately selected depending on the type of the cell extract and the preparation method.
  • a stabilizing component is added to the cell extract in various amounts and types on a trial basis, and after an appropriate time, a step of eliminating the low-molecular-weight synthesis inhibitor is performed. After that, the cell extract is separated into a soluble fraction and an insoluble fraction by a method such as centrifugation, and a method in which an insoluble substance is less generated is selected.
  • the obtained post-treatment cell extraction A method in which cell-free protein synthesis is performed using the effluent and a protein having high protein synthesis activity is selected is also preferable.
  • an appropriate stabilizing component is also added to an outer dialysis solution, and dialysis is performed using these components for an appropriate time.
  • substantially free of an insoluble substance refers to an insoluble substance to the same extent as a cell extract that has been subjected to the treatment for eliminating a low-molecular-weight synthesis inhibitor by the various methods described above. Means that the protein has been removed, and whether or not it has been removed can be confirmed by the level of its protein synthesis activity of the obtained cell extract.
  • the wheat germ extract and the dialysate have an ATP of 100 ⁇ to 0.5 mM, GTP of 25 tM to 1 mM,
  • Examples of the 0 kinds of amino acids include a method of adding 25 ⁇ M to 5 mM and performing dialysis for 30 minutes to 1 hour or more.
  • the temperature for performing dialysis may be any temperature as long as protein synthesis activity is not lost and dialysis can be performed.
  • the minimum temperature is the temperature at which the solution does not freeze, usually 10 ° C, preferably 15 ° C, and the maximum temperature is the temperature that does not adversely affect the solution used for dialysis.
  • the temperature is 40 ° C, preferably 38 ° C.
  • the method of adding the stabilizing component to the cell extract is not particularly limited, and it is added before the step of eliminating the low-molecular-weight synthesis inhibitor, incubated for an appropriate period of time, and allowed to stabilize.
  • a substance exclusion step may be performed, or a low-molecular-weight synthesis inhibitor is eliminated using a cell extract to which a stabilizing component has been added and / or a buffer to be used in the exclusion step to which a stabilizing component has been added.
  • a step may be performed.
  • the cell extract of the present invention preferably a cell extract after dialysis treatment, particularly preferably a cell extract substantially free of insoluble substances, can be stored in any state. It can be stored at 20 ° C or lower, more preferably at 180 ° C or lower.
  • a lyophilized state is particularly preferred.
  • a ready-made 5-cell extract it can be stored in a lyophilized state, and when used, protein synthesis can be performed only by dissolving and adding translation type-I. This is a more preferred embodiment.
  • the thus-obtained cell extract from which the low-molecular-weight synthesis inhibitor has been eliminated can be introduced into each of the selected systems or devices known per se to perform protein synthesis.
  • systems or devices for protein synthesis include Notte fe (Pratt, J. M. eta 1., Transcription and Transcription, Hames, 179—209, BD & Higgins, SJ, eds, IRLP ress. , Ox ford (1 984)) 5, and adding the amino acid or tRNA necessary for cell-free protein synthesis to the cell extract.
  • the reaction may be stopped if protein synthesis is performed for a long period of time5, and the reaction can be maintained for a long time by using the latter continuous or discontinuous supply system of amino acids and energy sources. And efficiency can be further improved.
  • the wheat germ extract was prepared by the blender method described in (1) above. In this case, tRNA is sufficiently contained, so that it is not usually necessary to add tRNA.
  • the above-mentioned synthesis reaction solution from which translation type III has been removed can be pre-incubated for an appropriate period of time if necessary, and then added with translation type III and incubated.
  • the pre-incubation is 10 to 40.
  • the incubation is carried out at 10 to 40 ° C, preferably at 18 to 30 ° C, more preferably at 20 to 26 ° C for 5 to 10 minutes.
  • the reaction time is the time until the reaction stops, but is usually about 10 minutes to 7 hours in the batch method.
  • the synthesis reaction solution is used as the inner solution of the dialysis, and the protein is synthesized using a device that is separated from the outer solution of the deposition by a dialysis membrane capable of mass transfer.
  • a dialysis membrane capable of mass transfer.
  • dialysis container examples include a container having a dialysis membrane attached to the bottom (Daiichi Kagaku Co., Ltd .: dialysis cup 12, 2000, etc.) and a dialysis tube (manufactured by Sanko Junyaku Co., Ltd .: 12, 2, 0, 0) Etc.).
  • a dialysis membrane one having a molecular weight limit of 100,000 daltons or more is used, and one having a molecular weight limit of about 12,200 daltons is preferable.
  • the dialysis external solution the one obtained by removing translation type I from the above synthesis reaction solution is used.
  • the reaction rate of the dialysate decreases, the dialysis efficiency can be increased by replacing it with fresh one.
  • the reaction temperature and time are appropriately selected depending on the protein synthesis system to be used, but are usually 10 to 40 ° C, preferably 18 to 30 ° C, more preferably 2 to 40 ° C in a system using a wheat germ extract. It can be performed at 0 to 26 ° C for 10 minutes to 12 days.
  • the synthesis reaction solution When performing protein synthesis using the overlay method, put the synthesis reaction solution into an appropriate container, and overlay the dialysis external solution described in the above-mentioned folding method on the solution without disturbing the interface. Performs protein synthesis. Specifically, for example, after pre-incubating the above-mentioned synthesis reaction solution excluding translation type II for an appropriate time as necessary, Then, put it in an appropriate container to obtain a reaction phase. Examples of the container include a microtiter plate and the like. The reaction is carried out by overlaying the outer layer of the dialysis (supply phase) described in the above dialysis method on the upper layer of this reaction phase without disturbing the interface.
  • the interface between the two phases does not necessarily have to be formed in a horizontal plane by means of a multilayer, and a horizontal plane can be formed by centrifuging a mixed solution containing both phases.
  • a horizontal plane can be formed by centrifuging a mixed solution containing both phases.
  • the diameter of the circular interface of both phases is 7 mm
  • the volume ratio between the reaction phase and the feed phase is suitably 1: 4 to 1: 8, but 1: 5 is preferred.
  • the larger the interface area consisting of both phases the higher the mass exchange rate due to diffusion, and the higher the protein synthesis efficiency. Therefore, the capacity ratio of both phases changes depending on the interface area of both phases.
  • the reaction temperature and time are appropriately selected depending on the protein synthesis system to be used under static conditions, but in a system using a wheat germ extract, the reaction temperature is 10 to 40 ° C, preferably 18 to 30 ° C. C, more preferably at 20-26 ° C, usually for 10-17 hours. When an E. coli extract is used, the reaction temperature is preferably 30 to 37 ° C.
  • the synthesis reaction is performed using the synthesis reaction solution, and when the synthesis reaction is stopped, type I RNA, amino acids, energy sources, etc. are supplied. Protein synthesis is performed by discharging the synthesized products and decomposed products. Specifically, for example, after pre-incubating the above-mentioned synthesis reaction solution excluding the translation type III for an appropriate time as required, the translation type III is added, and the mixture is placed in an appropriate container to carry out the reaction. Examples of the container include a microplate. Under this reaction, for example, in the case of a reaction solution containing a wheat germ extract at a volume of 48% by volume, the synthesis reaction is completely stopped in one hour. This was confirmed by measuring the incorporation of amino acids into proteins and analyzing polyribosomes by sucrose density gradient centrifugation (Proc. Natl. Ac Ad. Sc in USA, 97, 559-564 (2000)). You can check.
  • reaction solution after the synthesis reaction is stopped is passed through a gel filtration ram equilibrated in advance with a feed solution having the same composition as the dialysis external solution described in the above dialysis method.
  • a feed solution having the same composition as the dialysis external solution described in the above dialysis method.
  • This filtered solution is passed through a gel filtration ram equilibrated in advance with a feed solution having the same composition as the dialysis external solution described in the above dialysis method.
  • the synthesis reaction resumes, and protein synthesis takes several hours. Progress over time.
  • this reaction and the gel filtration operation are repeated.
  • the reaction temperature and time are appropriately selected depending on the protein synthesis system used. ⁇ In a system using a wheat germ extract, it is preferable to repeat gel filtration at about 26 ° C. about every hour.
  • the protein thus obtained can be confirmed by a method known per se. Specifically, for example, measurement of incorporation of amino acids into proteins, separation by SDS-polyacrylamide gel electrophoresis and staining with Coomassie brilliant blue (CBB), autoradiography method (Endo, Y. eta 1., J. Biotec h., 25, 221-230 (l 992); Proc. Natl. A0cad. Sci. US A., 97, 559-564 (2000))
  • the reaction solution thus obtained contains a high concentration of the target protein, it can be purified by a method known per se such as dialysis, ion exchange chromatography, affinity chromatography, or gel filtration.
  • the target protein '5 can be easily obtained from the reaction solution.
  • step (1) or (2) for eliminating the low-molecular-weight synthesis inhibitor and the step for stabilizing the cell extract in this step are performed by using all of the components required for cell-free protein synthesis other than the translation type II.
  • a ready-made cell extract can be prepared by performing the reaction in a solution containing All of the components required for cell-free protein synthesis other than translation type II differ depending on the origin of the cell extract used, and whether the cell-free protein synthesis of the present invention is a cell-free translation system. It also differs depending on whether it is a cell-free transcription / translation system.
  • the wheat germ cell-free translation system contains at least amino acid and energy sources (ATP and GTP) as substrates, and the cell-free transcription / 5 translation system contains Nucleic acid and RNA polymerase are required.
  • ATP and GTP amino acid and energy sources
  • Nucleic acid and RNA polymerase are required.
  • various ions, a buffer, an ATP regeneration system, and a nucleus It further includes one or more of an acid-degrading enzyme inhibitor, tRNA, a reducing agent, polyethylene glycol, 3,5′-cAMP, folate, and an antibacterial agent.
  • Specific concentrations of ATP are preferably 100 ⁇ to 0.5 mM
  • GTP is 25 ⁇ M to 1 mM
  • 20 kinds of amino acids are preferably 25 ⁇ to 5 ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ .
  • Each of the other five components is also included at a concentration commonly used in the art.
  • addition of tRNA is usually not necessary.
  • the cell extract for cell-free protein synthesis of the present invention preferably the cell extract after treatment, 10 and the ready-made cell extract, can be provided as a kit for cell-free protein synthesis reaction containing the same.
  • the kit of the present invention contains, in addition to the above, components essential or suitable for the protein synthesis system, specifically, amino acids serving as substrates. ,energy
  • RNA polymerase a positive control translation type I nucleic acid
  • a vector for producing translation type I a reaction vessel, and the like.
  • all components essential or suitable for the above-mentioned protein synthesis system are contained, so that other components are included. However, it is not necessary to include all these reagents, and any combination of reagents may be used as long as the kit can be used for cell-free protein synthesis.
  • the color sorter includes a means for irradiating the crude germ fraction with light, a means for detecting reflected light and Z or transmitted light from the crude germ fraction, a means for comparing the detected value with a reference value, and a means for deviating from the reference value.
  • This is a device that has a means for selecting and excluding objects that fall within the reference value.
  • the crude germ fraction was supplied on a belt of a color sorter so as to have a particle size of 1,000 to 5,000 Zm 2, and the reflected light was detected by irradiating the crude germ fraction on the belt with a fluorescent light.
  • the belt transport speed was 50m / min.
  • a monochrome CCD line sensor (2048 pixels) was used as the light receiving sensor.
  • a beige belt is attached to eliminate the black components (seed coat, etc.) from the germ, and a reference value is set between the germ luminance and the seed coat luminance, and those that deviate from the reference value are aspirated. Removed by Then, in order to sort the endosperm, it was replaced with a dark green belt, a reference value was set between the brightness of the embryo and the brightness of the endosperm, and those out of the reference value were removed by suction. Suction was performed using 30 suction nozzles (one suction nozzle per 1 cm in length) arranged about 1 cm above the conveyor belt.
  • embryos were selected until the purity of the embryos (weight ratio of embryos contained in 1 g of any sample) reached 98% or more.
  • the obtained wheat germ fraction is suspended in distilled water at 4 ° C and washed using an ultrasonic cleaner. The solution was washed until it no longer became cloudy. Next, 0.5 volume of Nodide (Nakarai, manufactured by Tectonitas) P40. The suspension was suspended in a / 0 solution and washed with an ultrasonic washer until the washing solution did not become cloudy to obtain a wheat germ. The following operation was performed at 4 ° C.
  • the filtrate was previously dissolved in a solution (4 OmM HEPE S-KOH, pH 7.8, 10 OmM potassium acetate, 5 mM magnesium acetate, 4 mM dithiothreitol, and 0.3 mM each of 20 L-type amino acids).
  • a solution (4 OmM HEPE S-KOH, pH 7.8, 10 OmM potassium acetate, 5 mM magnesium acetate, 4 mM dithiothreitol, and 0.3 mM each of 20 L-type amino acids).
  • Gel filtration is performed on a Sephadex G-25 column (Amersham Pharmacia Biotech) equilibrated with a mixture (neither adding an amino acid or a labeled amino acid depending on the purpose of protein synthesis). went.
  • the wheat germ extract prepared in Example 1 contains factors necessary for protein synthesis (tR NA, aminoacyl-tRNA synthetase, ribosome, translation initiation factor, peptide chain elongation factor, translation termination factor, etc.) and other molecules, especially substances that specifically or non-specifically inhibit protein synthesis Therefore, we attempted to prepare a wheat extract from which this was fractionated by utilizing the difference in molecular weight, and from which protein synthesis inhibitors were eliminated.
  • factors necessary for protein synthesis tR NA, aminoacyl-tRNA synthetase, ribosome, translation initiation factor, peptide chain elongation factor, translation termination factor, etc.
  • a dialysis membrane was used for fractionation.
  • the dialysis membrane used was Spectra / Pore 6 (S PECTRUM LABORATOR IES INC., CA'.USA) with an excluded molecular weight of 50,000. This excluded molecular weight did not pass through the factors necessary for protein synthesis, and was selected as being able to fractionate molecules having a molecular weight lower than that.
  • the wheat germ extract used for dialysis was extracted from a protein synthesis reaction solution containing no ATP, GTP, or amino acid (Extract 1 of wheat germ extract (2/3 of the total volume) in Example 1).
  • the SDS-PAGE analysis was carried out after collecting the dialysis solution by precipitation with 10% trichloroacetic acid. From this result (Fig. 2 (B)), a small amount of low-molecular-weight molecules with a molecular weight of about 14,000, which migrated to the tip of the gel, were detected in the dialysis solution after 512 hours.
  • the amount of protein in the dialysis solution calculated by the densitometer method was 7 ⁇ g (in the presence of ATP) and 23 / ig (ATP non-
  • the protein eliminated by the dialysis performed in (1) above does not show a significant difference in mass between the two dialysis conditions.
  • a reaction solution for protein synthesis containing the wheat germ extract-containing solution 61 prepared in the above dialysis in a dialysis membrane 25 ⁇ l (2 OmM5 HEPES-KOH ( ⁇ 7.6) at the final concentration respectively, 95 mM acetic acid) Potassium, 2.65 mM magnesium acetate, 0.38 OmM spermidine (manufactured by Nacalai Tectonitas), 20 mM each of 0.3 mM L-type amino acid, 4 mM dithiothreitol, 1.2 mM AT P (manufactured by Wako Pure Chemical), 0.25 mM GTP (manufactured by Wako Pure Chemical), 16 mM creatinine phosphate (manufactured by Wako Pure Chemical), lU // i 1 RNa sei nh ibiter (manufactured by TA KARA), 0 5 ⁇ g / 1 creatine kinase (Roche) and 1
  • the mRNA for translation type I (QGF P) is linked to the omega ( ⁇ ) sequence of tobacco mosaic virus (TMV), which is the translation initiation reaction sequence linked to the SP6 : mouth motor sequence, and further to the 3 'downstream.
  • TMV tobacco mosaic virus
  • Plasmid GF PZ p EU (WO 01/27260) containing GFP gene DNA was transcribed using SP6 RNA po 1 ymerase (manufactured by TOYOBO), and the resulting RNA was subjected to fermentation. After extraction with ethanol and ethanol, precipitation was performed with Nick Colum (Amersham Pharmacia Biotech) and used.
  • the wheat germ extract obtained by dialysis in the presence of ATP As for the charge (white graph), protein synthesis was promoted as the dialysis time was increased, and reached 236% of that of undialyzed (black graph) in 12 hours of dialysis.
  • the protein synthesis activity was significantly reduced, and it was found that after 12 hours of dialysis, it decreased to 37.5% of that of undialyzed ones.
  • Nucleic acid was extracted from the insoluble fraction obtained in Example 2 (2) above by the SDS-phenol method.
  • the method for extraction, quantification, and gel electrophoresis of RNA is described in a paper by Endo et al. (Endo, Y. eta 1., J. Biotec h., 25, 22 1-230 (1992)). The method described was used.
  • Fig. 4 shows the results.
  • the amount of nucleic acid is measured by the ultraviolet absorption value (A 260 nm).
  • the wheat germ extract obtained by dialysis in the presence of ATP was used (Figure 4A, graph shown in the garden), although only a small amount of nucleic acid was detected, but no ATP was present.
  • the one using the wheat germ extract obtained by the below analysis contained a high concentration of nucleic acid.
  • the insolubilization of the solution containing the nucleic acid lasted until 4 hours from the start of dialysis, and thus showed a constant value after 2 hours (see Fig. 1A).
  • Undialyzed sample lml contains about 10A260 nm units of tRNA, and transfer of less than 8% of tRNA to insoluble material reduces protein synthesis by 30% (6 hours analysis, Fig. 3)
  • the tRNA contained in this insoluble substance may be a specific molecular species that recognizes specific codons on mRNA.
  • ATP functions as an insolubilization inhibitor in the reaction for producing such insoluble substances including tRNA (+ ATP lane).
  • Intracellular tRNA and aminoacyl-tRNA do not exist in a free state, but form a complex structure with aminoacyl-tRNA synthetase, 5S rRNA and some ribosomal proteins (M. M irande, et al., Er. J. B ioc 3 ⁇ 4 em. 147, 281-289 (1 985); K. Og ataetal., J. B ioch em. , 1 10, 1037-1044 (1 991)). Taking these reports together and considering them further, the experimental results presented here suggest that ATP is involved in their complex formation. Therefore, whether or not the effect of preventing the insolubilization of the wheat germ extract during the dialysis and the activation effect of the protein synthesis activity is a phenomenon specific to ATP only was examined below.
  • Example 4 Examination of insoluble substance formation prevention factor (extract solution stability inducer) and protein synthesis inactivation inhibitory factor in exclusion of low molecular substances from wheat germ extract by dialysis membrane
  • Extract 1 described in (1) with 0.25 mM GTP added Extract 4 as extract 1 with 20 L-type amino acids (0.3 mM each) added, and extraction Liquid 5 was prepared by adding 1.2 mM ATP, 0.25 mM GTP, and 20 kinds of L-type amino acids (0.3 mM each) to Extract 1.
  • Example 4 (1) The wheat germ extract obtained in Example 4 (1) was used to synthesize GFP mRNA type II protein in the same manner as in Example 2 (3), and the amount of synthesized protein was measured in the same manner from the fluorescence intensity. did. The result is shown in Fig. 5 (B).
  • GTP (+ (GTP)) was found to promote protein synthesis almost as well as ATP (+ (ATP)), almost in agreement with the effect of preventing insolubilization.
  • the amino acid (+ (AAs')) exhibited a certain effect of preventing insolubilization of tRNA, the effect of promoting the activity of protein synthesis was hardly recognized.
  • Extract 4 described in Example 4 (1) (Wheat germ extract to which all factors required for protein synthesis were added: 1.2 mM ATP, 0.25 mM GTP, 20 types of L-type amino acids ( (0.3 mM each) was allowed to stand at 26 ° C, the same condition as for dialysis, for 6 hours, and protein synthesis was performed using GFP mRNA as type III in the same manner as in Example 2 (3). The amount of the synthesized protein was measured in the same manner from the strength.
  • Figure 6 shows the results. In the figure, the graph indicated by ⁇ indicates the result of the extract subjected to the above operation, and the graph indicated by ⁇ indicates the result of the extract not subjected to the above operation (conventional method).
  • the cell-free protein synthesis reaction using wheat germ extract has already been carried out in the presence of components necessary for protein synthesis such as ATP, GTP, and amino acids in batch reactions. It has been reported that the amount of protein synthesized can be significantly increased by performing the operation of standing at 30 ° C for 30 minutes according to the method (Y. Shuliangetal., J. Fermentent. B ioen g. 84, 548-552, (1 997)). Based on the results, they found that the effects of ATP, GTP, and amino acids were: (i) the initiation factor 2 'GTP.
  • Aminoacyl-tRNA triple complex (eIF_2'GTP' am inoacyl-tR (Ii) The generation and accumulation of (43) S preinitiation complex (43 S preinitiation complex).
  • the results shown in the above examples demonstrate that prolonged dialysis promotes protein synthesis activity even in the presence of ATP or GTP alone. It is known that the 43S pre-initiation complex formation reaction in the initial stage of protein synthesis requires GTP, but does not require ⁇ ⁇ ⁇ . Given the fact that the dialysis operation in the presence of ATP alone promotes protein synthesis activity shown in this experiment, our findings have already been reported (Y. Shui 1iang, eta 1., J. Fe 84, 548-552, (1997)), suggesting a different mechanism.
  • Example 4 (2) The temperature dependence of the effect of promoting protein synthesis activity by dialysis of the wheat germ extract confirmed in Example 2 (4) and Example 4 (2) was analyzed.
  • Extract 4 described in Example 4 (1) (wheat germ extract to which all factors necessary for protein synthesis were added. 1.2 mM ATP, 0.25 mM GTP S 20 L-type amino acids (0.3 mM each) was dialyzed for 6 hours at 26 ° C and 4 ° C, and protein synthesis was performed using GFP mRNA as type II in the same manner as in Example 2 (3). Similarly, the amount of the synthesized protein was measured.
  • a solution in which the extract 5 was allowed to stand at 4 ° C. for 6 hours was also used as a control experiment. Figure 7 shows these results.
  • the phenomenon of promoting protein synthesis activity is that low-molecular-weight translational inhibitors or non-specific interfering substances are present in the wheat germ extract, and these are excluded from the translation factor group by dialysis treatment. It was proved that this was caused by: On the other hand,
  • Dialysis at 4 ° C in the absence of ATP produces insoluble substances. This indicates that even at 4 ° C, ATP and the like are important for stabilizing translation factors, especially for long-term stabilization.
  • the reaction solution was 3 OmM HEPES-KOH, pH 7.6, 95 mM potassium acetate, 2.65 mM magnesium acetate, 2.85 mM dithiothreitol, 1.2 mM ATP, 0.25 mM GTP, 16
  • a solution containing mM creatine phosphate, 0.5 mg Zm 1 creatine kinase, 0.38 OmM spermidine, 20 L-type amino acids (0.3 mM each) 1000 units / m 1 ribonuclease inhibitor (RNa sin) was added as the GFP mRNA described in Example 2 '(4), and a synthesis reaction was performed at 26 ° C.
  • the horizontal axis in the figure is the volume of the added yellow V, low molecular fraction.
  • Protein synthesis activity was measured from radioactivity counts incorporated into [ 14C ] bite-isine protein at 2 hours at 26 ° C.
  • the radioactivity count incorporated into the protein in the reaction solution 51 was set to 3600 dpm as 100%. .
  • the protein synthesis inhibitors shown here are contained in the adsorbed fraction of the Sephadex G25 column, and are also excluded from Suptra / pore 6 or exclusion molecular weight of 500,000 daltons. Since some substances are excluded by the regenerated cellulose membrane having a molecular weight of 12,000 to 14,000 daltons, it is considered to be a substance having a molecular weight of less than about 14,000 daltons.
  • Example 7 Examination of Wheat Germ Extract Containing All the Components Necessary for Protein Synthesis Other than Type ⁇ -Wheat embryo cell-free protein synthesis reaction solution .
  • all these products contain wheat germ extract and a solution containing amino acids, energy sources, ions, etc. in separate containers. It had to be mixed with the te type. These mixing operations are complicated because they need to be performed at a low temperature of about 0 to 4 ° C, and have caused the synthesis reaction to fail.
  • the construction of such a protein synthesis reaction kit is not suitable for exhaustive synthesis of proteins from a large number of genes, and such complicated processes must be solved for fully automated robots in the future. It was one of the biggest challenges.
  • the extract 5 described in Example 4 (1) contains ATP, GTP, and amino acids in addition to the wheat germ extract.
  • Preparing wheat germ extract with enhanced synthetic ability ( Figure 5B). Therefore, if a sample is used in which the concentration of each component is adjusted to the optimal concentration for cell-free protein synthesis using a wheat germ extract prior to dialysis, mRNA is added to the solution after dialysis. Cell-free protein synthesis should be able to be carried out easily simply by manipulation.
  • Wheat germ extracts of three kinds of lots obtained according to the method of Example 1 were prepared to have the composition of extract 5 described in Example 4 (1), and were used in Example 2 (1
  • the dialysis was performed for 12 hours by the method described in (2).
  • the protein synthesis activity of dialyzed and undialyzed wheat germ extracts was determined by the continuous method using a dialysis membrane, using GFP mRNA as type II in the same manner as described in Example 2. (4). It was measured.
  • FIG. FIG. 9A shows the results of analysis of the sample 48 hours after the protein synthesis reaction by SDS-polyacrylamide gel electrophoresis.
  • the coomassie brilliant pull-single band indicated by the arrow is the GFP product.
  • the amount of GFP synthesized using the dialyzed wheat germ extract (dialysis (+)) is not dialyzed, regardless of the extract of any of the lots A, B, and C. It can be seen that the amount is clearly increased as compared with the wheat germ extract (dialysis treatment (1)).
  • the graph of FIG. 9B shows the amount of protein synthesized per 1 ml of the reaction solution calculated by densitometry based on the purified GFP preparation. It can be seen that the dialysis treatment (Sonoichi) has almost twice the synthesis capacity as that of the non-dialysis treatment (violent).
  • a low-molecular-weight protein synthesis inhibitor is eliminated from a cell extract used in a cell-free protein synthesis system, and a cell extract having enhanced protein synthesis activity can be obtained.
  • the step of removing the low-molecular inhibitor from the cell extract is performed in the presence of all of the components required for protein synthesis except for the translation type II, thereby providing a ready-made cell extract.

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Abstract

L'invention concerne un procédé de préparation d'un extrait cellulaire haute performance en vue d'effectuer une réaction de synthèse de protéine acellulaire, procédé comprenant l'élimination d'une substance à bas poids moléculaire ayant une activité inhibant la synthèse de protéine à partir d'un extrait cellulaire ayant une activité de synthèse de protéine acellulaire, par dialyse, filtration sur gel, ultrafiltration ou analogue. L'invention concerne en outre un extrait cellulaire tout préparé, en vue d'effectuer la synthèse de protéine acellulaire au moyen de cet extrait. En éliminant la substance à bas poids moléculaire inhibant la synthèse de protéine dans la co-existence de ATP, GTP ou d'un aminoacide, la formation d'une substance insoluble peut être empêchée et, de ce fait, l'extrait cellulaire peut être stabilisé.
PCT/JP2003/000975 2002-01-31 2003-01-31 Extrait cellulaire pour synthese de proteine acellulaire et procede de production de cet extrait WO2003064672A1 (fr)

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US10/503,259 US8734856B2 (en) 2002-01-31 2003-01-31 Cell extract for cell-free protein synthesis and process for producing the same
JP2003564263A JP3675804B2 (ja) 2002-01-31 2003-01-31 無細胞タンパク質合成用細胞抽出液、及びその製造方法
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WO2006043675A1 (fr) * 2004-10-22 2006-04-27 Cellfree Sciences Co., Ltd. Extrait de cellule pour synthèse protéique acellulaire et méthode de préparation dudit extrait
JPWO2004097014A1 (ja) * 2003-04-25 2006-07-13 株式会社セルフリーサイエンス ハイスループット合成システム及び該システムを自動で行うための合成装置
JP2012511145A (ja) * 2008-12-05 2012-05-17 キアゲン ゲーエムベーハー ホルマリン固定組織からの種々の生体分子の並行抽出

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EP1477566B1 (fr) * 2002-01-31 2007-09-26 CellFree Sciences Co., Ltd. Extrait de germes permettant la synthese acellulaire de proteines et procede permettant de produire ces dernieres
WO2005063979A1 (fr) * 2003-12-26 2005-07-14 Cellfree Sciences Co.,Ltd. Extrait cellulaire pour la synthese proteique acellulaire a haute fonctionnalite et procede de preparation dudit extrait
US20140193852A9 (en) * 2004-12-23 2014-07-10 Erik Vossman Adipose tissue collection and pre-processing devices for use in liposuction procedure
US20120301904A1 (en) 2011-04-26 2012-11-29 Prosetta Antiviral, Inc Multiprotein assemblies
EP2927312B1 (fr) * 2012-11-30 2022-10-19 Bioneer Corporation Appareil de préparation automatique de protéines sans cellule et procédé de préparation de protéines utilisant celui-ci
CN104497101B (zh) * 2015-01-16 2017-11-03 北京七巧时代科技有限公司 一种小麦胚芽细胞活性蛋白的制备方法
WO2019035916A1 (fr) 2017-08-15 2019-02-21 Northwestern University Conception de sites de glycosylation protéiques par expression et caractérisation rapides des n-glycosyltransférases
US11673921B2 (en) 2017-11-10 2023-06-13 Northwestern University Cell-free protein synthesis platform derived from cellular extracts of Vibrio natriegens
WO2019204346A1 (fr) 2018-04-16 2019-10-24 Northwestern University Procédés pour co-activer in vitro l'incorporation et la glycosylation d'acides aminés non standard (nsaa) dans des lysats cellulaires bruts
US11814621B2 (en) 2018-06-01 2023-11-14 Northwestern University Expanding the chemical substrates for genetic code reprogramming

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JP4643444B2 (ja) * 2003-04-25 2011-03-02 株式会社セルフリーサイエンス ハイスループット合成システム及び該システムを自動で行うための合成装置
WO2006043675A1 (fr) * 2004-10-22 2006-04-27 Cellfree Sciences Co., Ltd. Extrait de cellule pour synthèse protéique acellulaire et méthode de préparation dudit extrait
JP2012511145A (ja) * 2008-12-05 2012-05-17 キアゲン ゲーエムベーハー ホルマリン固定組織からの種々の生体分子の並行抽出

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